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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
21. to 24. March 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Remazol Schwarz B Gran.

Test animals

Species:
hamster, Chinese
Strain:
other: Han: Chin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zentralinstitut fur Versuchstiere, Hannover
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: males: mean = 29.2 g (25 - 33 g)
females: mean = 27.29 (24 - 31 g)
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: no data
- Housing: one animal per cage
- Diet: Altromin 7010 hamster diet (Altromin GmbH, Lage/lippe), ad libitum
- Water: tap water in plastic bottles ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 21. to 24. March 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: sesame oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 50% suspension in sesame oil


Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
12, 24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5000 mg/kg body weight
Basis:
other: nominal dose
Remarks:
Doses / Concentrations:
500 mg/mL
Basis:
nominal conc.
No. of animals per sex per dose:
Control and Reactive Black 5: 5 males and 5 females per killing time point
Positive control: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (Endoxan: batch 05459)
- Justification for choice of positive control(s): availability of historical data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg body weight (5 mg/mL)

Examinations

Tissues and cell types examined:
bone marrow cells from femur: 50 metaphases/animal
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: MTD based on preliminary study


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered orally by gavage to the test animals at a single dose of 5000 mg/kg bodyweight. Sesame oil was administered in the same way to the negative control group. A positive control group, introduced after exactly 24 hours to run parallel with the negative control and the dose groups, received Endoxan at an oral dose of 50 mg/kg bodyweight. Two hours before killing by carbon dioxide asphyxation (12, 24, or 48 hours after treatment ), the hamsters each received an intraperitoneal injection of 3.3 mg demecolcin (Colcemid) per kg bodyweight.

DETAILS OF SLIDE PREPARATION:
After killing, both femora were removed and the bones completely stripped of muscle tissue. After removal of the epiphyses, the bone marrow was flushed in alternate directions out of the diaphysis into a centrifuge tube by means of a syringe containing Hanks solution (2 ml/femur) at the temperature of 37°C, mixed and centrifuged for 5 minutes at 1000 rpm after which all but a few drops of the supernatant was drawn off by pipette and sediment resuspended by tapping.
For hypotonic treatment, approximately 5 ml of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37°C. Addition of 1.5 ml fixative (methanol : glacial acetic acid 3 + 1) and flow through with air. After re-centrifugation for five minutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 ml fixative. After 20 minutes the fixation was removed carefully with a pipette and suspended in 2.5 ml fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4°C.
After re-centrifuging for 5 minutes at 1000 rpm, all but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a Pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4°C, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2% orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan or Eukitt


METHOD OF ANALYSIS:

After the slides had been coded, 50 metaphases per animal were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified. The metaphases were examined for the following aberrations: gap (g), break (b), fragment (f), minute (m), deletion (d), exchanges including intrachanges (ex), dicentricity (di), chromosome disintegration (cd) ring (ri) and polyploidy (pp). In addition, metaphases with 5 and more aberrations were classified separatly as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared at each killing time with the results from the dose groups and the positive control.

Evaluation criteria:
1. Structural aberrations
Gap: Non stained segment (achromatic gap) of chromatide without dislocation of the apparently separate part, irrespective of size of the non-stained area.
Break: A visible fracture of the chromatide structure where the broken piece is laterally dislocated or shifted in the longitudinal axis but can still be assigned to the corresponding centric part.
Fragment: Acentric part of a chromosome which may appear individually, regardless of their size.
Minute: Small chromatide body with a diameter smaller than the width of the chromatide.
Deletion: Terminal or interstitial losses of part of the chromatide.
Exchange: These are exchange aberrations, subdivided into intrachanges (the union of parts that can combine, within a chromosome) and interchanges (the union of parts that can combine from two or more chromosomes). Dicentric chromosomes and ring chromosomes are included in this group.

The chromatide aberrations specified above can also occur as iso-chromatide aberrations (e.g. isochromatid break)

2. Numerical aberrations
Aneuploidy: A deviation from the typical number of individual chromosomes in a set of chromosomes; a decrease in the number is known as hypoploidy an a increase as hyperploidy.
Polyploidy: More than two sets of chromosomes.

3. Additional criterion:
Chromosomal disintegration: where all or most of the chromosomes are irregular particles. If exchange figures occur in the metaphases, they are only included in this aberration group.
Statistics:
Comparison of no of aberrations of treated and control groups

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
feces black colored; urine greyblue colored
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw (highest applicable dose)
- Clinical signs of toxicity in test animals: no clinical signs, no deaths
- Evidence of cytotoxicity in tissue analyzed: -


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: not different from vehicle control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Remazol-Schwarz B Gran. is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.
Executive summary:

Remazol-Schwarz B Gran. was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose.

A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight.

Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. 5 males and 5 females from each group were killed at each of these times.

The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, Remazol-Schwarz B Gran. caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group.

Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, Remazol- Schwarz B Gran. is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.